表达人IL-17F重组腺病毒载体的构建及其表达产物的功能研究

Construction of recombinant adenovirus vector expressing hIL-17F and functional study of expressed IL-17 F

目的构建表达人白细胞介素IL-17F （hIL-17F）的重组腺病毒载体（Ad-hIL-17F），为进行hIL-17F基因表达抑制血管形成和抑瘤效应的研究奠定基础。方法以pUCm-T/hIL-17F重组质粒为模板PCR扩增hIL-17F，酶切连接到带有GFP标记的pAdTrack-CMV转移质粒上，PmeⅠ线性化重组质粒pAdTrack-CMV-hIL-17F，与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌，经同源重组，获得同源重组腺病毒质粒pAdEasy-1-pTrack-CMV-hIL-17F经PacⅠ线性化后转染QBI-293A细胞，收获Ad-hIL-17F，用RT-PCR和间接免疫荧光法鉴定人IL-17F基因表达。 MTT法检测hIL-17F基因表达对ECV304细胞的生长抑制作用， ELISA法检测人血管内皮生长因子（ VEGF）和血管生成素（ Ang-1）基因在293A细胞、ECV304细胞中的表达水平；实时定量RT-PCR检测Ad-hIL-17F对293A细胞中人VEGF转录的影响。结果测序显示hIL-17F序列正确，RT-PCR和间接免疫荧光法检测到了IL-17 F基因的表达。 Ad-hIL-17 F能显著抑制ECV304细胞的生长，抑制人VEGF和Ang-1基因在293 A细胞、ECV304细胞中的表达。结论成功构建和获得了hIL-17F的重组腺病毒载体（Ad-hIL-17F）， Ad-hIL-17 F可通过下调VEGF和Ang-1的分泌而抑制血管形成。

Objective To construct a recombinant adenovirus vector （ Ad-hIL-17F） expressing human interleukin 17F （hIL-17F） and to investigate the effects of expressed hIL-17F on angiogenesis. Methods The hIL-17F fragments was amplified by PCR using pUCm-T/hIL-17F plasmids as templates and then cloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-hIL-17F.The pAdTrack-CMV-hIL-17F transfer vector was linearized with PmeI digestion and then transformed into competent BJ 5183 with pAdEasy-1 backbone vector for homologous recombination .Then it was linearized with PacI digestion and transfected into human embryonic kidney 293 （QBI-293A） cells to construct Ad-hIL-17F.RT-PCR analysis and indirect immunofluorescent assay （IFA） were performed to determine the expressions of hIL-17F.MTT assay was used to detect the inhibitory effects on cell growth of ECV 304 .The expressions of VEGF and Ang-1 in 293 A and ECV304 cells were measured by ELISA .The effects of Ad-hIL-17 F on the expressions of VEGF in 293A cells were analyzed by Real-Time PCR.Results The sequencing result verified that hIL-17F gene fragment was correctly inserted in the vector .The expressions of hIL-17F gene at mRNA and pro-tein levels were confirmed by RT-PCR and IFA.Ad-hIL-17F could significantly inhibit the growth of ECV304 cells and down-regulate the expressions of VEGF and Ang-1 in 293A and ECV304 cells.Conclu-sion Ad-hIL17F expressing hIL-17F was successfully constructed .The expressed hIL-17F could inhibit the angiogenesis through down-regulating the expressions of VEGF and Ang-1.